1. Field of the Invention
This invention pertains to the field of the purification of polynucleotides from mixtures that contain fluorescent dye-labeled molecules. The polynucleotides can include, for example, reaction products from nucleic acid sequencing reactions.
2. Background
The demands of the Human Genome Project and the commercial implications of polymorphism and gene discovery have driven the development of significant improvements in DNA sequencing technology. Contemporary approaches to DNA sequencing have imposed stringent demands on reliability and throughput for DNA sequencers. Recent reports have demonstrated the extraordinary potential of capillary electrophoresis (CE) for DNA sequencing given the inherent speed, resolving power and ease of automation associated with this method as compared to slab gel electrophoretic methods (Carrilho et al., Anal. Chem. 1996, 68, 3305-3313; Tan and Yeung, Anal. Chem. 1997, 69, 664-674; Swerdlow et al., Anal. Chem. 1997, 69, 848-855).
Relative to cross-linked gel capillary electrophoretic columns, the recent development of replaceable polymer solutions to achieve size separation of single-stranded DNA fragments has increased the lifetime of the columns and eliminated the requirements of gel pouring and casting (Ruiz-Martinez et al., Anal. Chem. 1993, 65, 2851-2858). Additionally, improvements in the composition of the separation matrix have led to sequencing over 1000 bases per run (Carrilho et al., Anal. Chem. 1996, 68, 3305-3313). Automated capillary electrophoresis systems for DNA sequencing have been introduced commercially by three major scientific instrument manufacturers (Beckman Coulter CEQ(trademark) 2000 DNA Analysis System; Amersham Pharmacia MegaBACE 1000 DNA Sequencing System; and PE Biosystems ABI Prism 3700 DNA Analyzer).
Realizing the potential of this new generation of automated DNA sequencers is proving difficult, however, as problems in read length and accuracy remain, primarily due to the limitations associated with the methods currently available for purifying the products of cycle sequencing reactions. Indeed, the critical importance of sample preparation for the successful implementation of capillary electrophoresis has not been sufficiently emphasized.
In contrast to slab gel electrophoresis, primer extension products are introduced into the capillary column using electrokinetic injection, which provides focusing of the single-stranded DNA fragments at the head of the column (Swerdlow et al., Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 9660-966). However, electrokinetic injection is biased toward high electrophoretic mobility ions, such as chloride, deoxynucleotides and dideoxynucleotides, which, if present in the sequencing reaction solution, negatively affect the focusing of single-stranded DNA fragments. Consequently, to increase the amount of DNA injected into the capillary column, and to improve the focusing of the injected DNA, an effective removal of these small ionic species is required.
A further group of especially problematic high electrophoretic mobility ions are the dye-labeled fluorescent dideoxynucleotide terminators, and in particular the recently commercialized terminators having two fluorescent moieties configured as energy transfer pairs (e.g., ABI PRISM BigDye(trademark) Terminators from PE Biosystems and DYEnamic ET(trademark) Terminators from Amersham Pharmacia). These reagents, and the hydrolysis products derived therefrom, have been found to be particularly difficult to remove from primer extension reactions, resulting in the presence of fluorescent artifacts (routinely described as xe2x80x9cdye blobsxe2x80x9d) that negatively affect the automated analysis of sequencing data (Rosenblum, B.; Lee, L.; Spurgeon, S.; Khan, S.; Menchen, S.; Heiner, C. and Chen, S., Nucleic Acids Research, 1997, 25, 4500-4504). Dye-labeled sequencing primers, when employed as an alternative to dye-labeled terminators, afford similar problems (Jingyue, J.; Ruan, C.; Fuller, C.; Glazer, A. and Mathies, R., Proc. Natl. Acad. Sci. USA, 1995, 92, 4347-4351. Jingyue, J.; Kheterpal, I.; Scherer, J.; Ruan, C.; Fuller, C.; Glazer, A. and Mathies, R., Anal. Biochem., 1995, 231, 131-140. Jingyue, J.; Glazer, A. and Mathies, R., Nucleic Acids Research, 1996, 24, 1144-1148. Lee, L.; Spurgeon, S.; Heiner, C.; Benson, S.; Rosenblum, B.; Menchen, S.; Graham, R.; Constantinescu, A.; Upadhya, K. and Cassel, J., Nucleic Acids Research, 1997, 25, 2816-2822).
Fluorescent energy transfer dye-labeled dideoxynucleotide triphosphate terminators suitable for use in DNA sequencing are described in U.S. Pat. No. 5,800,996. Fluorescent energy transfer dye-labeled primers suitable for use in DNA sequencing are described in U.S. Pat. Nos. 5,688,648, 5,707,804 and 5,728,528.
During the course of cycle-sequencing reactions, deoxynucleotide triphosphates (dNTPs) and dye-labeled dideoxynucleotide triphosphates (ddNTPs) undergo hydrolysis of the phosphate ester bonds during the denaturation step that proceeds each amplification cycle when the temperature is elevated to from 95xc2x0 C. to 99xc2x0 C. This results in the generation of dye-labeled artifacts including dideoxynucleotide diphosphates (ddNDPs), dideoxynucleotide monophosphates (ddNMPs) and dideoxynucleosides. The ddNTPs derived from the pyrimidine bases, dideoxythymidine (ddTTP) and dideoxycytidine (ddCTP), are particularly labile in this regard. The dye-labeled ddNTPs, ddNDPs and ddNMPs elute from capillary electrophoretic columns prior to the dye-labeled primer extension products, and consequently do not directly interfere with the interpretation of the sequencing data. However, the intensity of the signals associated with the dye-labeled ddNTPs, ddNDPs and ddNMPs may exceed those of the primer extension products by many orders of magnitude. This discontinuity in signal intensity has proven problematic with respect to automated base-calling software, resulting, in a worse-case scenario, in the software interpreting the fluorescent artifact peaks as primer extension products and the primer extension products as baseline noise. A further significant complication is associated with the presence of dye-labeled dideoxynucleosides, in that they are found to co-elute from both capillary electrophoretic columns and slab gels with the primer extension products. Not only is the intensity of the artifact signal disproportionately high as compared to the signals associated with the primer extension products, but the width of the peak is sufficiently broad to obscure the analysis of from 5 to 20 bases.
The sample preparation scheme now routinely employed for both slab gel electrophoresis and CE consists of desalting DNA sequencing samples by ethanol or isopropanol precipitation, followed by reconstitution of the DNA fragments and template in a mixture of formamide-0.5 M EDTA (49:1) prior to loading or injection (Figeys et al., 1996, 744, 325-331; Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y., 1989; section 9.49). Although widely utilized, this method has been found to exhibit variable reproducibility in terms of DNA recovery, to provide marginal performance with respect to the quantitative removal of dye-labeled artifacts, and is not easily automated (Tan, H.; Yeung, E. S. Anal. Chem. 1997, 69, 664-674, and Hilderman, D.; Muller, D. Biotechniques 1997, 22, 878-879).
High electrophoretic mobility ionic species in DNA sequencing samples are not the only contaminants that cause degradation in sequencing read length. Template DNA also has been shown to interfere with the analysis of primer extension products in both thin slab gels (Tong et al., Biotechniques 1994, 16, 684-693), and capillary columns (Swerdlow et al., Electrophoresis 1996, 17, 475-483). Upon injection of the sequencing reaction solution, a current drop and significant deterioration in the resolving power of the capillary column is observed when template DNA is present in the sample (Salas-Solano et al., Anal. Chem. 1998, 70, 1528-1535). However, at present, template DNA removal is seldom considered an essential aspect of sample preparation for DNA sequencing by capillary electrophoresis.
Two approaches to sample preparation that address the need for removal of template and other reactants have been proposed thus far. In the first approach, which is described in U.S. Pat. No. 5,484,701, a biotinylated primer enables the capture and purification of primer extension products on streptavidin magnetic particles. After extensive washing of the primer extension products immobilized on the streptavidin magnetic particles to remove the sequencing reaction constituents including template DNA and unincorporated deoxynucleotides and dideoxynucleotides, release of the primer extension products from the particles is effected by heating the streptavidin magnetic particles to from about 90xc2x0 C. to 100xc2x0 C. in a formamide solution.
Although this approach has considerable utility in conjunction with slab gel electrophoresis (in which formamide is often added to sequencing samples to facilitate denaturation of duplex DNA and to increase the viscosity of the sample to facilitate slab gel loading), it has recently been shown to be problematic when utilized in conjunction with capillary electrophoresis. At least three distinct problems (exclusive of cost) have been identified as being associated with this approach. First, the formamide solution utilized to effect release of immobilized primer extension products is incompatible with electrokinetic injection, owing to the high ionic strength of the solution due to the presence of high electrophoretic mobility ions (most notably 10 mM EDTA or 30-140 mM sodium acetate in 95% formamide). In the absence of salt in the formamide solution, the efficiency of release of biotinylated primer extension products has been shown to be significantly reduced from  greater than 95% to  less than 40% (Tong and Smith, Anal. Chem. 1992, 64, 2672-2677). The effective ionic strength of the release solution has been shown to be still further increased by decomposition of 95% formamide that occurs when the solution is heated and results in release of ammonia.
Second, samples recovered from streptavidin magnetic particles are found to be contaminated with protein derived from streptavidin. Release of immobilized primer extension product results from the denaturation of the streptavidin that is covalently linked to the magnetic particle. Streptavidin is a multi-subunit protein with a high isoelectric point. Denaturation of immobilized streptavidin is always accompanied by the concomitant release of those protein subunits that are not covalently linked to the magnetic particles. This contaminating protein acts in a manner somewhat analogous to template DNA, as a consequence of its anionic character and high molecular weight. Finally, dye-labeled fluorescent dideoxynucleotide terminators, and hydrolysis products derived therefrom, have been found to bind nonspecifically to streptavidin magnetic particles, and to be released into the formamide solution upon denaturation of streptavidin. Thus, the nonspecifically bound terminators can accompany the xe2x80x9cpurifiedxe2x80x9d primer extension product and adversely impacting the subsequent analysis.
A second approach to purification of primer extension products that includes removal of template DNA and unincorporated reactants utilizes a multi-step methodology involving: (1) Ultrafiltration to remove template DNA; (2) Vacuum concentration to reduce sample volume; (3) Size exclusion chromatography (two sequential gel filtration columns) to reduce the ionic strength and remove dye-labeled artifacts; and (4) Vacuum concentration to reduce sample volume prior to analysis (Ruiz-Martinez et al., Anal. Chem. 1998, 70, 1516-1527, and Salas-Solano et al., Anal. Chem. 1998, 70, 1528-1535). Although this approach affords excellent samples for CE analysis, it is generally complex, costly, time consuming and unsuitable for automation in a high throughput environment. In fact, as compared to the throughput potential of multi-column capillary electrophoresis DNA sequencers, the aforementioned methodology would constitute the rate-limiting step in a sequencing laboratory.
The methods currently employed to purify primer extension products for analysis by capillary and slab gel electrophoresis have been summarized above. Although ethanol or isopropanol precipitation are known to reduce the level of both high electrophoretic mobility ions and dye-labeled artifacts present in the sample, the residual level of dye-labeled artifacts remains problematic. Size exclusion chromatography in spin columns can remove both high electrophoretic mobility ions and dye-labeled artifacts, but is the least automation compatible for high throughput environments and the most expensive. Additionally, the use of spin columns results in considerable sample dilution, often introducing the necessity for a precipitation or evaporation step to concentrate the sample prior to analysis. The biotin/streptavidin methodology should, in principle, quantitatively remove dye-labeled artifacts. However, dye-labeled artifacts bind nonspecifically to immobilized streptavidin and are released along with the primer extension products when the sample is heated in formamide. Another disadvantage of the biotin/streptavidin methods is that short molecules are preferentially adsorbed to the matrix, resulting in a decrease in signal for longer molecules.
Thus, none of the methods currently available provide for the quantitative removal of all of the potentially contaminating constituents associated with DNA sequencing reactions. Consequently, a method is needed to circumvent this considerable limitation if the extraordinary potential of capillary electrophoresis for DNA sequencing is to be realized in the not too distant future. The present invention fulfills this and other needs.
The present invention provides methods for removing fluorescent or other dye-labeled molecules from a mixture that includes one or more polymers, such as polynucleotides, into which dye-labeled molecules are incorporated as well as the unincorporated dye-labeled molecules. The methods involve contacting the mixture with a plurality of particles that are composed of a cross-linked three-dimensional hydrophilic polymeric matrix that has trapped within it a porous adsorptive hydrophobic material. The unincorporated dye-labeled molecules pass through the hydrophilic matrix and become preferentially adsorbed onto the porous hydrophobic materials. Subsequent removal of the particles from the mixture thus also removes the adsorbed dye-labeled molecules from the mixture that includes the polymers.
In some embodiments, the invention provides methods for the purification of fluorescently labeled primer extension products derived from polymerase chain reactions (PCR) and cycle sequencing reactions (CSR). The primer extension reaction mixtures generally contain unincorporated dye-labeled primers and/or terminators, including dye-labeled energy transfer primers and terminators (ddNTPs), as well as their hydrolysis products, which include dye-labeled ddNDPs, ddNMPs and dideoxynucleosides. The methods afford samples that are substantially free of fluorescent artifacts and thus are suitable for DNA sequencing by either capillary or slab gel electrophoresis.
The methods of the invention involve, in some embodiments, extending a primer by means of a template-directed primer extension reaction in the presence of either dye-labeled primers or dye-labeled terminators;
contacting the constituents of the primer extension reaction with a plurality of particles that are composed of a cross-linked three-dimensional hydrophilic polymeric matrix in which porous adsorptive hydrophobic materials are entrapped, so as to effect the preferential absorption to the particles of the dye-labeled primers or dye-labeled terminators, as well as dye-labeled artifacts derived therefrom; and
physically separating the particles from the liquid phase that contains the remaining constituents of the primer extension reaction.
Some embodiments of the methods of the invention further involve purifying the liquid phase that contains the remaining constituents of the primer extension reaction, if necessary, and analyzing the liquid phase that contains the remaining constituents of the primer extension reaction by capillary or slab gel electrophoresis.
The invention also provides methods for preparing dye-labeled polynucleotides that are substantially free of unincorporated dye-labeled reactants. These methods involve:
(a) annealing a primer to a template and contacting the annealed primer with a polymerase in a reaction mixture that comprises the dye-labeled reactant, thereby extending the primer to form a plurality of dye-labeled polynucleotides;
(b) contacting the reaction mixture with a plurality of particles that are composed of porous hydrophobic materials that are retained within a hydrophilic polymeric matrix, so as to effect the preferential absorption of the unincorporated dye-labeled reactant, and dye-labeled artifacts derived therefrom; and
(c) separating the particles of (b) from the reaction mixture that contains the dye-labeled polynucleotides.
Other aspects of the invention include, for example, the use of blocking reagents to precondition the magnetic particles so as to minimize the nonspecific binding of primer extension products.